QIAseq cfDNA All-in-One Kits

For conversion of cfDNA from plasma to NGS library for any liquid biopsy samples

Optimal conversion of cfDNA at every step from plasma to NGS library through highly efficient ligation chemistry

Go directly from eluant to library prep without quantification using a protocol supporting the widest range of cfDNA input (1–100ng)

Generate PCR-free libraries from 10 ng of cfDNA

Minimize PCR bias by the use of high-fidelity amplification reagents

Reduce cross-contamination risk with pre-dispensed plate-based adapters for up to 96 samples/NGS run

QIAseq cfDNA All-in-One Kits provide an optimized solution for cfDNA analysis using any NGS platform from Illumina or Ion Torrent. Kits enable optimal conversion of cfDNA at every step – from plasma to NGS library – and allowsensitive detection of even the rarest variants, providing you with the highest confidence in your results.

QIAseq cfDNA All-in-One Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Designed for any NGS-based cfDNA research.

Up to 5 ml plasma can be used for cfDNA extraction using QIAamp Mini Columns and 24 samples are processed in less than 2 h. For library preparation, 1–100 ng of cfDNA can be directly used from up to 56.5 µl eluant volume. Quantification of cfDNA prior to library prep is not necessary. Library prep includes end-polishing and adapter ligation, and takes <1 h. Optional HiFi amplification can be performed for <10 ng cfDNA before samples undergo sequencing.

Superior conversion rate of cfDNA molecules to NGS library.

Using either the QIAseq cfDNA All-in-One Kit or a kit from Supplier N, 8 cfDNA samples (input 20–37 ng in a total volume of 43 μl) were used for library construction. The average calculated conversion rate of the replicate samples is displayed. The QIAseq cfDNA All-in-One Kit shows significantly higher conversion rates.

Low error rates and high accuracy in workflows with and without PCR.

A. PCR-free library preparation using the QIAseq cfDNA All-in-One Kit was performed using 4 independent replicates of the same circulating cfDNA sample with an input amount of 9.35 ng. Even with inputs of less than 10 ng, the QIAseq cfDNA library prep reagents achieved consistent library yields higher than 2 nM. B. A comparison of normalized coverage distribution for libraries generated from cfDNA samples using the QIAseq cfDNA All-in-One Kit shows no significant differences in coverage for the workflow with or without PCR.

Equivalent results with manual and automated extraction.

As part of the QIAseq cfDNA All-in-One workflow, circulating cfDNA was isolated using manual QIAamp or automated QIAsymphony protocols. Library prep was performed using 1 ng isolated cfDNA (in 4 replicates), followed by sequencing. A. The QIAseq cfDNA library prep delivers library concentrations that are comparable to those achieved for cfDNA isolated with the QIAamp and QIAsymphony protocols and shows very low sample-to-sample variation. B. Sequencing quality metrics (represented here by the percentage of unique reads) are very good for library preparations from cfDNA isolated with the QIAamp and QIAsymphony protocols as part of the QIAseq cfDNA All-in-One workflow.

Reference samples of cfDNA with known mutations were subjected to the QIAseq cfDNA workflow. Sensitive detection of variants down to even 1% allelic frequency was found. The expected allelic frequencies from Horizon Discovery were used as the standard.

Performance

Designed for cfDNA

The QIAseq cfDNA All-in-One protocol is specially optimized for cfDNA and generates sequencer-ready NGS libraries from plasma in less than 3 h (see figure "Designed for any NGS-based cfDNA research"). It uniquely combines cfDNA extraction from plasma with library preparation chemistry in a way that avoids sample loss, while ensuring optimal sample conversion at every step. This results in very uniform whole genome or whole exome coverage (see figure "Highly uniform coverage").

Minimal bias through PCR library amplification

Enzyme formulations and buffers optimized for cfDNA yield sufficient library concentrations and allow PCR-free preparation of NGS libraries that are compatible with Illumina sequencers from just 10 ng of isolated cfDNA (see figure "Low error rates and high accuracy in workflows with and without PCR. If starting with less than 10 ng cfDNA, libraries can be amplified using the HiFi PCR master mix, a high-fidelity master mix that minimizes PCR bias, also included with the kit.

For library preparation for Ion Torrent sequencers, we recommend library amplification using the HiFi PCR master mix (included with the kit) prior to sequencing regardless of the sample input amount to enrich for correct adapter-ligated library molecules.

Flexibility with cfDNA extraction method without loss of samples or reduction in data quality

The QIAseq cfDNA All-in-One workflow consists of two major steps: extraction of cfDNA from plasma and library preparation for NGS.

cfDNA extraction

The QIAseq cfDNA Extraction Kit uses QIAamp Mini Columns to isolate cfDNA from up to 5 ml of plasma. The protocol is specially optimized for NGS with regards to the sensitivity required for variant detection. Following the typical “lyse, bind, wash, elute” steps, cfDNA is first released from histones and selectively bound to the silica membrane. Other contaminants, inhibitors and proteins in the plasma are washed out. In the final step, cfDNA is released from the membrane and eluted in a buffer provided with the kit.

As cfDNA is typically around 170 bp in size, fragmentation prior to library preparation is not required. cfDNA from 1–100 ng in up to 43 µl or 56.5 µl eluant can be directly used for Illumina or Ion Torrent library preparation, respectively.

Library preparation for Illumina sequencers

Library preparation for Illumina sequencers follows a simple one-tube, two-step procedure. In the first step, the ends of the cfDNA are repaired and A-tailed at the 3’ end. This allows the ligation of Illumina-specific sequencing adapters in the second step. The sequencing adapters included with the QIAseq cfDNA All-in-One Kit are dual-barcoded and are provided in a 96-well sealed plate. This allows multiplexing up to 96 different samples in a single sequencing run. Very limited amounts of cfDNA input of less than 10 ng can be amplified using the high-fidelity PCR master mix included with the kit.

Library preparation for Ion Torrent sequencers

Library preparation for Ion Torrent sequencers uses a proprietary All-in-One reaction that combines end-polishing and adapter ligation. End-repair and adapter ligation are performed in one step in a single tube. QIAseq sequencing adapters provided in the kit are dissolved in duplex buffer and are ready to use. Each well of the 24-plex single-use plate contains equimolar mixes of universal and individual barcode adapters. This allows multiplexing up to 24 different samples in a single sequencing run. Only the adapters supplied with the kit are compatible with the innovative All-in-One end repair and ligation reaction. The kit also contains high-fidelity amplification reagents and primers that are specific for Ion Torrent sequencers to enrich for correct adapter-ligated cfDNA molecules.

Applications

The QIAseq cfDNA All-in-One Kit can be used for any NGS-based cfDNA application. It generates NGS libraries from plasma for any whole genome or hybrid capture sequencing application and is thus suitable for biomarker discovery research, cancer therapy monitoring or non-invasive prenatal testing.